Table 1.
Measurement of translocation ATPase activity with bacterially expressed [3H]-prSSU as substrate
| Experiment | Signal ATPase activity (nmol ATP hydrolyzed ⋅mg Chl−1⋅h−1) | Import rate (pmol prSSU imported ⋅mg Chl−1⋅h−1) | Translocation ATPase activity (ATP hydrolyzed per prSSU imported) |
| 1 | 131.0 | 125.0 | 1048 |
| 2 | 66.7 | 79.0 | 844 |
| 3a | 48.7 | 73.1 | 666 |
| 4b | 26.4 | 30.9 | 854 |
| 5 | 56.5 | 76.8 | 736 |
| 6 | 70.2 | 88.6 | 792 |
| 7 | 91.1 | 92.0 | 990 |
| Average | 847 ± 135 |
Chloroplasts were incubated at room temperature in the dark for 30 min in IB containing 5 μM tentoxin and 10 μM DCMU. ATP mixed with [γ-32P]-ATP was then added to the chloroplasts reaching a final concentration of 1 mM, which was then incubated in the dark at room temperature for 15 min. DTT was added to final concentration of 1 mM (except experiment 3). Import reactions were started by adding 42.6 pmol of bacterially expressed [3H]-prSSU (final concentration, 0.71 μM) or the resulting flow-through from concentrating [3H]-prSSU using Ultra-15 Centrifugal Filtration Devices (Amicon, cutoff 10 kDa). Samples were withdrawn at 0 and 30 min. Reactions assayed for ATPase activity were stopped by adding an ice-cold acidic charcoal solution (2% Norit charcoal, 0.25 M HCl, 0.25 M sodium pyrophosphate, and 0.25 M K2HPO4,) After separating Pi from ATP and other nucleotides using charcoal (Materials and Methods), released [32P] was quantified by scintillation counting. Identical samples analyzed for protein import were stopped by adding 600 μL ice-cold IB; chloroplasts were isolated immediately by centrifugation. The amount of imported SSU was analyzed by fluorography and quantified using ImageQuant. a[DTT] was 0.27 mM. b5 μM triphenyltin was included in the chloroplast pretreatment together with tentoxin and DCMU.